Sustained PIP3 production generates transient Rac activation. (A) dHL-60 cells expressing our optogenetic system for controlling PI3K were treated with either 10 nM fMLP (black curve) or red light (red curve) for 10 min. PIP3 levels in each sample were measured using MS. Chemoattractant drove transient increases in PIP3, whereas opto-PI3K drove sustained increases in PIP3. Each point represents a mean of three independent experiments. Error bars indicate SD. (B, top) Cells were placed under inactivating IR light for 5 min followed by exposure to activating red light for 10 min. Rac* was isolated from total cell lysate at indicated time points via Pak pulldown and then quantified via immunoblot using an antibody targeting total Rac. (Middle) Cells were stimulated with 1,000 nM fMLP for 10 min. Samples were collected, processed, and analyzed as in the top panel. Both opto-PI3K and chemoattractant drove transient activation of Rac. (Bottom) Representative immunoblots of total Rac for experiments shown in top and middle panels. For top and middle panels, each point represents a mean of three independent experiments. (C and D, top) Cells were placed under inactivating IR light for 5 min followed by exposure to activating red light for 10 min (C) or treatment with 10 nM fMLP (D). Antibodies targeting phospho-Akt (S473), phospho-Pak, and total Pak were used as PIP3 readouts, Rac* readouts, and loading controls, respectively. The kinetics of these indirect readouts were consistent with the kinetics of our direct readouts of PIP3 and Rac*. Each point represents a mean of three independent experiments. Error bars indicate SEM. (C and D, bottom) Representative immunoblots. Molecular masses are given in kilodaltons.
